Transcript Document

Evolution and synthetic biology
Eörs Szathmáry
Collegium Budapest
Eötvös University
To find out why life is as it is
• Optimal solurion among alternatives
• Historical contigency (lack of alternatives
at the time of evolution)
• Vestigial traits (adaptive only in the past,
but now frozen)
• Understanding by doing (engineering—the
airplane MUST fly)
• Compare with synthetic organic chemistry
The ways of synthetic biology
• DNA technology (modify natural systems with
some synthetic components)
• To perform something natural with unnatural
organic components
• At the extreme to create unnatural chemical
systems with biological properties
• Unnatural assembly of natural modules
(repressilator)
The extended genetic alphabet
Why is aminoA not used?
The importance of repeating charge
Artificial synthesis of the new base
pair dZ:dP
• Several polymerase enzymes accept it
• Can be used in PCR reactions
New base pair in the code for new
amino acid
• Implementation with
isoC:isoG pair
• UAG nonsense codon for
iodotyrosine
• Or the (iso-C)AG codon
• Challenge: coupling of
non-standard amino acids
to nonstandard tRNAs by
nonstandard synthetases
Fisher’s (1930) question
"No practical biologist
interested in sexual
reproduction would be led to
work out the detailed
consequences experienced by
organisms having three or
more sexes; yet what else
should he do if he wishes to
understand why the sexes
are, in fact, always two?"
•We must go back to the RNA world
•RNA was both enzyme and genetic
material
•The alphabet was optimal then!
•More than 3 billion years ago…
Some RNA molecules act as
enzymes (ribozymes) today
The fitness of ribo-organisms
W(N) = A(N) Q(N)
(vö. Eigen, 1971)
W
fitness
A
reproduction rate
Q
fidelity of replication
N
size of the alphabet
W(N) = A(N) Q(N)
There is somewhere an optimal N
Von Kiedrowski’s replicator
Peptide replicator networks
Towards Systems Chemistry
• Analysis of synthesis of coupled
autocatalytic chemical systems
• Origin of life and chemical technology
E - FLUX
Fet Open
Contract n° FP7-225167
History III: Gánti and fluid automata
Pathways of supersystem evolution
metabolism
MB
boundary
MT
template
BT
MBT
INFRABIOLOGICAL SYSTEMS
The stochastic corrector model
for compartmentation
Szathmáry, E. &
Demeter L. (1987)
Group selection of early
replicators and the
origin of life. J. theor
Biol. 128, 463-486.
Grey, D., Hutson, V. &
Szathmáry, E. (1995) A
re-examination of the
stochastic corrector
model. Proc. R. Soc.
Lond. B 262, 29-35.
E - FLUX
Fet Open
Contract n° FP7-225167
History II: The stochastic corrector
model and Andrew’s letter
Theory
Experiment
In vitro compartmentalization
E - FLUX
Fet Open
Contract n° FP7-225167
Integrated digital microfluidic system for directed evolution
Droplet production
Droplet reinjection
Droplet incubation
Co-encapsulation of gene
and amplification system
Gene amplification
Droplet pairing
Fluorescence detection
Droplet sorting
Pair gene containing droplet
(small droplet) with in vitro
transcription/ribozyme
substrate mixture (large
droplet)
Activity
Droplet fusion
No
activity
Detection of
ribozyme
activity and
selection of
droplets
exceeding
desired threshold
Droplet incubation
Ribozyme expression and activity
E - FLUX
Fet Open
Contract n° FP7-225167
Model system: ribozyme RNase X motif
Fluorescence-based assay and real time monitoring
+ X motif
Products
Atto488
Quencher
Substrate
- X motif
X motif
Cleavage
Orthogonal systems
Substrate A
X motif A
Substrate B
X motif B
Substrate C
X motif C
E - FLUX
Fet Open
Contract n° FP7-225167
Qb replicase amplification and ribozyme activity
Purification and activity of the Qb replicase
Crude extract
Qb replicase
Activity test
Purification
Amplified
MDV RNA
b-subunit
EF-Tu
EF-Ts
Qb-X motif A
X motif embedded in MDV retains its activity
Substrate A
Product A
Towards...
• Division of labour between genes and
enzymes
• Origin of chromosomes by spontaneous
recombination
• Unforeseen solutions by the system?
Szostak’s vesicles